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Chloranthones A–D: Minor and Unprecedented Dinor-Eudesmenes fromChloranthus elatior

by Shu-Ting Liua)b), Juan Xiong*a), Yu Tanga), Wen-Xuan Wanga)b), Van-Binh Buib),Guang-Lei Maa), Ya Huanga), Yun Zhaob), Guo-Xun Yanga), and Jin-Feng Hu*a)

a) Department of Natural Products Chemistry, School of Pharmacy, Fudan University, No. 826Zhangheng Rd., Shanghai 201203, P. R. China

(phone/fax: þ86-21-51980172; e-mail: [email protected]; [email protected])b) Department of Natural Products for Chemical Genetic Research, Key Laboratory of Brain Functional

Genomics, Ministry of Education, East China Normal University, No. 3663 North Zhongshan Rd.,Shanghai 200062, P. R. China

Four novel naturally occurring diastereoisomers of dinor-eudesmenes, named chloranthones A–D(1–4, resp.), were isolated as minor components from the EtOH extract of the aerial parts ofChloranthus elatior. The unprecedented framework was established using extensive 2D-NMR tech-niques. Their absolute configurations were deduced from the observed Cotton effects in their circulardichroism (CD) spectra. A plausible biosynthetic pathway to the dinor-eudesmenes is proposed.

Introduction. – Within our studies directed toward the discovery of novel bioactivesesquiterpenoids from higher plants [1] [2], the EtOH extract of the aerial parts of theperennial plant Chloranthus elatior Link (family Chloranthaceae) has been recentlyreinvestigated, and a number of eudesmanes were characterized [3]. We herein reporta further study on this plant, describing the isolation and structure elucidation of fourminor dinor-eudesmene metabolites, 1 – 4 (Fig. 1).

Results and Discussion. – The air-dried and powdered aerial parts of C. elatior wereextracted with 95% EtOH at room temperature, and the percolated extract wasconcentrated in vacuo. The crude extract was suspended in H2O and then exhaustivelyextracted with AcOEt. The AcOEt fraction was successively subjected to repeatedcolumn chromatography (CC) on silica gel, and semi-preparative HPLC to afford fourdiastereoisomers 1 – 4 (Fig. 1) in very limited amounts (ca. 1 mg of each from 6.4 kg ofthe dried plant material).

Chloranthone A (1) was obtained as colorless oil. The IR spectrum of 1 showedabsorption bands attributed to OH (3444 cm�1), ketone (1716 cm�1), and a,b-unsaturated ketone (1669 cm�1) groups. The presence of the latter functional moiety

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)904

� 2014 Verlag Helvetica Chimica Acta AG, Z�rich

Fig. 1. Chemical structures of 1–4

was supported by the absorption maximum at 248 nm (log e¼3.4) in the UV spectrum.The 1H-NMR spectrum (in C5D5N; Table 1) of 1 displayed three Me singlets (d(H)0.98 (Me(12)), 1.94 (vinylic Me(13)), 2.17 (Me(11))), and signals of one olefinic H-atom (d(H) 5.94 (br. s, H�C(3))) and one D2O-exchangable H-atom (d(H) 6.60 (s,HO�C(5))). These NMR data were similar to those of the known eudesmanolideschlorantholide F [4] and chlorelactone B [5], previously isolated from the same plant(C. elatior), implying that 1 might be also a eudesmane-type sesquiterpene. However,the 13C- and DEPT-NMR spectra of 1 only exhibited 13 signals corresponding to threesp3 Me, three sp3 CH2, one sp3 and one sp2 (d(C) 126.6) CH groups, and two sp3 (one O-bearing at d(C) 80.3) and three sp2 (d(C) 160.3, 200.1, 209.1) quaternary C-atoms(Table 2). The alleged dinor-sesquiterpenoid structure was confirmed by its molecularformula C13H18O3, which was determined based on a pseudo-molecular-ion peak at m/z245.1137 ([M þ Na]þ ) in its positive-ion-mode HR-ESI-MS.

The above data suggested that 1 was a dinor-eudesmene, the constitution of whichwas ascertained by extensive 2D-NMR experiments (Fig. 2). First, two spin systems(�CH2(6)CH(7)CH2(8)�,�CH¼CMe (allylic coupling between H�C(3) and Me(13)))were observed in the COSY-NMR spectrum of 1. One-bond H-atom-C-atomconnectivities and the long-range H,C couplings were then deduced from HSQC-and HMBC-NMR experiments. Clear nJ (n¼2, 3) correlations were observed betweenC(1) (d(C) 46.2) and H�C(3) (d(H) 5.94) and Me(12) (d(H) 0.98), between C(5)(d(C) 80.3) and CH2(6) (d(H) 2.06, 2.59), CH2(8) (d(H) 1.81, 2.37), and Me(13) (d(H)1.94), between C(9) (d(C) 48.5) and CH2(6) and CH2(8), between C(10) (d(C) 209.1)and H�C(7) (d(H) 3.16), CH2(8), and Me(11) (d(H) 2.17), as well as between C(12)(d(C) 24.0) and CH2(8) (Fig. 2). These correlations revealed that 1 was a dinor-eudesmene with a degraded five-membered ring B.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014) 905

Table 1. 1H-NMR Data of Compounds 1–4a). d in ppm, J in Hz. Atom numbering as indicated in Fig. 1.

H-Atom 1b) 2b) 2c) 3b) 4b)

Ha�C(1) 3.24 (d, J¼16.8) 2.74 (d, J¼16.1) 2.47 (d, J¼16.6) 2.66 (d, J¼15.9) 3.25 (d, J¼16.4)Hb�C(1) 2.42 (d, J¼16.8) 2.39 (d, J¼16.1) 2.12 (d, J¼16.6) 2.41 (d, J¼15.9) 2.46 (d, J¼16.4)H�C(3) 5.94 (br. s) 5.98 (br. s) 5.76 (br. s) 5.99 (br. s) 5.93 (br. s)Ha�C(6) 2.59 (dd,

J¼13.6, 2.9)2.57 (dd,J¼14.1, 9.2)

2.29 (dd,J¼14.1, 9.2)

2.29 (dd,J¼14.6, 11.2)

2.21 (dd,J¼13.0, 8.9)

Hb�C(6) 2.06 (dd,J¼13.6, 11.3)

2.47 (dd,J¼13.9, 9.0)

2.38 (dd,J¼13.9, 9.0)

2.83 (dd,J¼14.4, 5.2)

2.45 (dd,overlapped)

H�C(7) 3.16 (m) 3.48 (m) 3.40 (m) 3.16 (m) 3.54 (m)Ha�C(8) 2.37 (dd,

J¼11.5, 9.6)1.91 (dd,J¼13.4, 4.4)

1.79 (dd,J¼13.4, 4.8)

1.79 (dd,J¼12.5, 9.0)

2.42 (dd,overlapped)

Hb�C(8) 1.81 (dd,J¼11.5, 8.9)

2.20 (dd,J¼13.4, 11.7)

2.13 (dd,J¼13.4, 12.1)

2.26 (dd,J ¼12.4, 9.3)

1.91 (dd,J¼12.0, 3.0)

Me(11) 2.17 (s) 2.11 (s) 2.18 (s) 2.14 (s) 2.17 (s)Me(12) 0.98 (s) 1.23 (s) 1.04 (s) 1.24 (s) 0.95 (s)Me(13) 1.94 (br. s) 2.10 (br. s) 2.04 (br. s) 2.08 (br. s) 1.96 (br. s)5-OH 6.60 (s) 6.61 (s) not detected 6.32 (s) 6.89 (s)

a) Assignments were achieved by a combination of 1D- and 2D-NMR experiments. b) Recorded inC5D5N (400 MHz). c) Recorded in CD3OD (500 MHz).

The relative configuration of 1 was established by the NOE correlations in itsNOESY-NMR spectrum (Fig. 2). The following clear NOE correlations wereobserved: Me(12) (d(H) 0.98)/Hb�C(1) (d(H) 2.42); Me(12)/H�C(7) (d(H) 3.16);Me(12)/Hb�C(8) (d(H) 1.81); H�C(7)/Hb�C(6) (d(H) 2.06); H�C(7)/Hb�C(8);HO�C(5) (d(H) 6.60)/Ha�C(1) (d(H) 3.24); HO�C(5)/Ha�C(6) (d(H) 2.59); andHa�C(6)/Ha�C(8) (d(H) 2.37). Based on these data and on biogenetic considerations,Me(12) and H�C(7) both adopted the b-orientation, whereas the tertiary OH at C(5)and the Ac at C(7) were a-oriented, as depicted in Fig. 2.


Table 2. 13C-NMR Data of Compounds 1–4a)

C-Atom 1b) 2b) 2c) 3b) 4b)

C(1) 46.2 49.9 50.2 48.6 46.5C(2) 200.1 197.5 200.8 197.7 199.9C(3) 126.6 125.7 126.0 125.4 126.5C(4) 160.3 163.4 166.2 163.8 159.9C(5) 80.3 82.8 83.9 81.8 81.3C(6) 38.6 40.5 40.9 40.7 34.2C(7) 48.1 48.1 48.8 48.4 48.1C(8) 33.2 39.9 40.6 39.9 36.9C(9) 48.5 50.1 51.0 50.6 47.4C(10) 209.1 209.2 212.1 208.8 209.1C(11) 28.1 28.9 29.1 28.3 28.6C(12) 24.0 20.9 20.7 20.5 24.6C(13) 19.5 19.9 19.9 19.9 19.3

a) Assignments were achieved by a combination of 1D- and 2D-NMR experiments. b) Recorded inC5D5N (100 MHz). c) Recorded in CD3OD (125 MHz).

Fig. 2. COSY Correlations and key HMBCs of 1, and key NOE correlations of 1–4

Chloranthones B, C, and D (2 –4, resp.) were all found to have the same molecularformula (C13H18O3) as that of 1 as deduced from their HR-ESI-MS data. Their IR, UV,and NMR (Tables 1 and 2) data closely resembled those of 1, implying they had thesame constitutional formulae. Comparing the 1H-NMR data of 2 with those of 1, signalsof H�C(7) and Me(12) of 2 were shifted downfield to d(H) 3.48 and 1.23, respectively,due to the pyridine-induced deshielding effects [6], requiring that HO�C(5), H�C(7),and the angular Me(12) in 2 assumed the same b-orientation, which was confirmed bykey NOE correlations Me(12) (d(H) 1.23)/H�C(7) (d(H) 3.48) and Me(12)/HO�C(5)(d(H) 6.61) in the NOESY spectrum of 2 (Fig. 2). Therefore, compound 2 was deducedto be the 5-epimer of 1. Accordingly, compound 3 was successively elucidated to be the7-epimer of 2, and compound 4 was the 5-epimer of 3, according to the pyridine-induced deshielding effects and the observed NOE correlations (Fig. 2).

Chloranthones A– D (1– 4, resp.) represent the four possible diastereoisomersresulting from the stereogenic C-atoms C(5) and C(7). Their absolute configurationswere deduced from observed Cotton effects in their circular dichroism (CD) spectra.The change in sign of the Cotton effects for the p�p* transition of the conjugated enonedue to the different configuration at C(5) was observed in compounds 1 – 4 (Fig. 3).The CD spectra of 1 (De247 �14.2) and 4 (De246 �6.1) exhibited diagnostic negativeCotton effects as in the case of a-rotunol [7], 5a-hydroxyisopterocarpolone [8], andkandenol B [9]. Thus, the configuration at C(5) in compounds 1 and 4 was assigned as(S) (5a-OH). On the contrary, both compounds 2 (De251 þ6.4) and 3 (De247 þ28.3)showed positive Cotton effects as described before for b-rotunol [7] and sibiriolides[10], resulting from a 5b-OH orientation (5R).

The rare chloranthones A– D (1 – 4, resp.) have a C13 skeleton previously unknownfor natural metabolites. Actually, obtaining such minor secondary metabolites andelucidating their structures require more time and effort when utilizing conventional[1– 3] [6] rather than high-throughput natural-product chemistry methods previouslyperformed by one of the authors [11 – 14]. Eudesmane-type sesquiterpenoids have beenpreviously reported from C. elatior [3– 5], which could be linked to the possible


Fig. 3. CD Curves of 1–4

formation of the new compounds 1– 4. Biogenetically, eudesmanes are generallyderived from farnesyl diphosphate (FPP) [15]. The dinor-eudesmenes 1– 4 with adegraded ring B are probably biosynthesized by a series of oxidation, degradation, andrearrangement that leads to the anticipated configuration (Scheme).

Similar to other eudesmanes isolated from C. elatior [3], compounds 1 –4 have beenevaluated for their cytotoxicities against human A549 and U-2OS cancer cells, theantioxidant effects on H2O2 production in H9c2 cardiac muscle cells, and the anti-inflammatory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) produc-tion in both RAW 264.7 and BV-2 cells, but they were found inactive.

This work was supported by NSFC grants (No. 81273401, 81202420), a STCSM grant (No.11DZ1921203), grants from the Ph.D. Programs Foundation of Ministry of Education (MOE) of China(No. 20120071110049, 20120071120049), and a MOST grant (No. 2011ZX09307-002-01).

Experimental Part

General. For instrumentation and general methods, see [3].Plant Material. The aerial parts of C. elatior were collected in Chongzhou, Sichuan Province, P. R.

China, by one of the authors (S.-T. Liu) in October 2010. The plant was identified by Prof. Bao-KangHuang (Second Military Medical University, Shanghai, P. R. China). A voucher specimen (No. JSL-01)was deposited with the Herbarium of the School of Pharmacy, Fudan University, Shanghai, P. R. China.

Extraction and Isolation. The dried, powdered aerial parts of C. elatior (6.4 kg) were extracted with95% EtOH (4�15 l) at r.t. to give a black crude extract (1.2 kg), which was suspended in H2O (2 l) andthen extracted with AcOEt (1.5 l�3). After removal of the solvent under reduced pressure, the AcOEtextract was subjected to CC (SiO2; petroleum ether (PE)/AcOEt 4 : 1 to 0 : 1) to afford four fractions,Frs. 1–4. Fr. 1 was subjected to CC (SiO2; PE/AcOEt 4 : 1 to 2 :1) to yield three subfractions, Frs. 1.1–1.3). Fr. 1.2 was separated by semi-prep. HPLC (MeCN/H2O 20 : 80 (v/v); flow rate, 3.0 ml/min) to yieldtwo minor components: 1 (tR 7.7 min; 1.3 mg) and 3 (tR 5.7 min; 1.1 mg). Similarly, compounds 2 (tR

9.4 min; 1.1 mg) and 4 (tR 11.4 min; 0.9 mg) were obtained from Fr. 1.3 (MeCN/H2O 25 : 75 (v/v); flow


Scheme. A Hypothetical Biosynthetic Pathway of 1–4

rate, 3.0 ml/min). In fact, 13 regular sesquiterpenoids including eudesmanes (e.g., shizukolidol andserralactone A) have been previously obtained from Fr. 1.1, Fr. 2, and Fr. 3 [3]. The non-sesquiterpenoid-containing fraction (Fr. 4) was discarded.

(5S,7S,9S)-Chloranthone A (¼ (2S,3aS,7aS)-2-Acetyl-1,2,3,3a,4,7a-hexahydro-7a-hydroxy-3a,7-di-methyl-5H-inden-5-one ; 1). Colorless oil. [a]20

D ¼ þ57.3 (c¼0.30, MeOH). UV (MeOH): 248 (3.4).CD (c¼1.4�10�3

m, MeOH): 247 (�14.2). IR: 3444 (br.), 1716, 1669, 1652, 1384, 1023. 1H- and13C-NMR: see Tables 1 and 2, resp. HR-ESI-MS: 245.1137 ([M þ Na]þ , C13H18NaOþ

3 , calc. 245.1148).(5R,7S,9S)-Chloranthone B (¼ (2S,3aS,7aR)-2-Acetyl-1,2,3,3a,4,7a-hexahydro-7a-hydroxy-3a,7-di-

methyl-5H-inden-5-one ; 2). Colorless oil. [a]20D ¼ �26.4 (c¼0.28, MeOH). UV (MeOH): 251 (3.4). CD

(c¼1.3�10�3m, MeOH): 251 (þ6.4). IR: 3422 (br.), 1711, 1672, 1654, 1384, 1051. 1H- and 13C-NMR:

Tables 1 and 2, resp. HR-ESI-MS: 245.1149 ([M þ Na]þ , C13H18NaOþ3 , calc. 245.1148).

(5R,7R,9S)-Chloranthone C (¼ (2R,3aS,7aR)-2-Acetyl-1,2,3,3a,4,7a-hexahydro-7a-hydroxy-3a,7-di-methyl-5H-inden-5-one ; 3). Colorless oil. [a]20

D ¼ þ28.6 (c¼0.28, MeOH). UV (MeOH): 247 (3.4). CD(c¼1.3�10�3

m, MeOH): 247 (þ28.3). IR: 3441 (br.), 1713, 1670, 1654, 1384, 1024. 1H- and 13C-NMR:Tables 1 and 2, resp. HR-ESI-MS: 245.1156 ([M þ Na]þ , C13H18NaOþ

3 , calc. 245.1148).(5S,7R,9S)-Chloranthone D (¼ (2R,3aS,7aS)-2-Acetyl-1,2,3,3a,4,7a-hexahydro-7a-hydroxy-3a,7-di-

methyl-5H-inden-5-one ; 4). Colorless oil. [a]20D ¼ �26.1 (c¼0.23, MeOH). UV (MeOH): 250 (3.5). CD

(c¼1.0�10�3m, MeOH): 246 (�6.1). IR: 3440 (br.), 1708, 1673, 1652, 1384, 1032. 1H- and 13C-NMR:

see Tables 1 and 2, resp. HR-ESI-MS: 245.1157 ([M þ Na]þ , C13H18NaOþ3 , calc. 245.1148).

REFERENCES

[1] V.-B. Bui, S.-T. Liu, J.-J. Zhu, J. Xiong, Y. Zhao, G.-X. Yang, G. Xia, J.-F. Hu, Phytochem. Lett. 2012,5, 685.

[2] X.-W. Li, L. Weng, X. Gao, Y. Zhao, F. Pang, J.-H. Liu, H.-F. Zhang, J.-F. Hu, Bioorg. Med. Chem.Lett. 2011, 21, 366.

[3] J. Xiong, S.-T. Liu, Y. Tang, W.-X. Wang, V.-B. Bui, Y. Zhao, H. Fan, G.-X. Yang, J.-F. Hu,Phytochem. Lett. 2013, 6, 586.

[4] F. Wang, D.-S. Zhou, G.-Z. Wei, F.-C. Ren, J.-K. Liu, Phytochemistry 2012, 77, 312.[5] C.-L. Sun, H. Yan, X.-H. Li, X.-F. Zheng, H.-Y. Liu, Nat. Prod. Bioprospect. 2012, 2, 156.[6] Y. Zang, J. Xiong, W.-Z. Zhai, L. Cao, S.-P. Zhang, Y. Tang, J. Wang, J.-J. Su, G.-X. Yang, Y. Zhao, H.

Fan, G. Xia, C.-G. Wang, J.-F. Hu, Phytochemistry 2013, 92, 137.[7] H. Hikino, K. Aota, D. Kuwano, T. Takemoto, Tetrahedron 1971, 27, 4831.[8] J.-F. Hu, S.-P. Bai, Z.-J. Jia, Phytochemistry 1996, 43, 815.[9] L. Ding, A. Maier, H.-H. Fiebig, W.-H. Lin, G. Peschel, C. Hertweck, J. Nat. Prod. 2012, 75, 2223.

[10] X.-Q. Zhang, W.-C. Ye, R.-W. Jiang, Z.-Q. Yin, S.-X. Zhao, T. C. W. Mak, X.-S. Yao, Nat. Prod. Res.2006, 20, 1265.

[11] J.-F. Hu, E. Garo, H.-D. Yoo, P. A. Cremin, L. Zeng, M. G. Goering, M. O�Neil-Johnson, G. R.Eldridge, Phytochem. Anal. 2005, 16, 127.

[12] J.-F. Hu, H.-D. Yoo, C. T. Williams, E. Garo, P. A. Cremin, L. Zeng, H. C. Vervoot, C. M. Lee, S. M.Hart, M. G. Goering, M. O�Neil-Johnson, G. R. Eldridge, Planta Med. 2005, 71, 176.

[13] H.-D. Yoo, P. A. Cremin, L. Zeng, E. Garo, C. T. Williams, C. M. Lee, M. G. Goering, M. O�Neil-Johnson, G. R. Eldridge, J.-F. Hu, J. Nat. Prod. 2005, 68, 122.

[14] J.-F. Hu, E. Garo, H.-D. Yoo, P. A. Cremin, M. G. Goering, M. O�Neil-Johnson, G. R. Eldridge,Phytochemistry 2005, 66, 1077.

[15] F. Yu, R. Utsumi, Cell. Mol. Life Sci. 2009, 66, 3043.

Received July 22, 2013
